The pancreas contains exocrine glands that secret digestive enzymes into the duodenum and the pancreatic islets which are endocrine glands. A treatment whereby the pancreatic islets, which play an important role in regulation of blood glucose concentration, are separated from the pancreas, purified, and transplanted to patients with insulin-dependent type 1 diabetes mellitus and the like is referred to as pancreatic islet transplantation. Because the pancreatic islets can be infused into the body in a manner of drip infusion, the pancreatic islet transplantation is minimally invasive and physical burden on the patient is lower.
Meanwhile, the exocrine gland accounts for about 90% of the pancreas; and technique for separating the pancreatic islets are difficult ones and exhibit an extremely low success rate. Currently, as an enzyme agent for cell and tissue dissociation, a mixture of subtypes called collagenase H (ColH) and collagenase G (ColG), both of which are derived from Clostridium histolyticum, is used for separation of the pancreatic islets from the pancreas in a practical clinical setting. However, because the tissue composition of the pancreas varies in age and degree of ponderal index, the separation of the pancreatic islets succeeds only when an enzyme agent for the pancreatic islet separation is compatible with pancreatic tissues provided.
The above ColH and ColG are multidomain proteins having plural domain structures and the activity thereof is associated with a combination and relative arrangement of the domains (Non Patent Literature 1). According to Non Patent Literature 1, collagenases derived from the genus Clostridium contain, in common, three domains namely a catalytic domain (hereinafter, may also be referred to as CD), a polycystic kidney disease-like domain (hereinafter, may also be referred to as PKD), and a collagen-binding domain (hereinafter, may also be referred to as CBD). ColH has a domain structure that is composed of one CD, two PKDs, and one CBD and represented by CD-PKD-PKD-CBD; and ColG has a domain structure that is composed of one CD, one PKD, and two CBDs and represented by CD-PKD-CBD-CBD. Non Patent Literature 1 discloses that calcium binds to the N terminal side of CBD and the N terminal portion of the domain undergoes structural change when calcium comes off; that calcium is important for collagen binding; that calcium contributes to stabilization of ColH; and the like.
Meanwhile, as for a microorganism-derived collagenase with a high specific activity, a Grimontia-hollisae-derived collagenase is available (Non Patent Literature 2). It is a collagenase that contains a prepro region, a catalytic domain, a linker region, and a prepeptidase C terminal domain (hereinafter, also referred to as PPC) and has a molecular weight of 84 kDa; and the result of BLAST search has indicated that it exhibits a low homology with ColH and ColG. Further, it is difficult to inexpensively obtain the Grimontia-hollisae-derived collagenase; and in light of this problem, a method of developing technique for production of such a collagenase using a genetic engineering technique has also been suggested (Patent Literature 1). This Patent Literature 1 describes a method of preparing a bacmid pCC1BAC-2 that contains a gene for the entire coding region of Grimontia-hollisae-derived collagenase and a method of preparing a Brevibacillus choshinensis recombinant using the bacmid pCC1BAC-2 to thereby produce the Grimontia-hollisae-derived collagenase.